Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.
...
PMID:The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase. 287 76

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.
...
PMID:Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules. 287 26

Subunit c of the membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli has been isolated under nondenaturing conditions (Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515-518) and antibodies have been raised in rabbits. The primary antisera did not recognize the antigen when present in the same buffer as used for the immunization. Surprisingly, in one of the three antisera a strong antibody binding was observed when intact F0, a.c complex or reconstituted subunit c was provided as the antigen. Incorporation of subunit c into liposomes together with subunits a and b forming an active, H+-translocating complex was not required for the recognition by the antiserum. Subunit c prepared by chloroform/methanol extraction or by chromatography in the presence of sodium dodecyl sulfate was not recognized by the anti-c antiserum when incorporated into liposomes.
...
PMID:Conformation-specific antiserum raised against subunit c of ATP synthase (F1F0) from Escherichia coli. 287 91

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.
...
PMID:Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase. 287 81

One subunit of the membrane portion of yeast ATP synthase was purified. Structural data are reported. This subunit (subunit 4) is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its apparent relative molecular mass is about 25,000. The polypeptide was extracted from the complex with a mixture of chloroform/methanol (1/1) and 0.5 M pyridinium acetate pH 6.0. Purification was performed with a combination of gel permeation chromatography on Sephadex G-75 and high-performance gel permeation chromatography with aqueous solvents containing 5% sodium dodecyl sulfate. The amino acid composition is reported here. The following sequence of the NH2-terminal ten residues was determined: Met-Ser-Ser-Thr-Pro-Glu-Lys-Gln-Thr-Asp.
...
PMID:Subunit 4 of ATP synthase (F0F1) from yeast mitochondria. Purification, amino-acid composition and partial N-terminal sequence. 288 7

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.
...
PMID:Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b. 288 81

Studies were carried out to determine whether a simple electron-dense "heavy atom" like iodine could be introduced selectively into one or more of the subunits of the mitochondrial ATP synthase complex of rat liver. Surprisingly, very low amounts of iodine are incorporated into the isolated F1 moiety of this complex under conditions which result in a marked loss of catalytic activity. ATPase activity is inactivated in a concentration-dependent manner at pH 7.5 with half-maximal inactivation occurring at about 40 microM iodine. A maximum of only 10 atoms of iodine are incorporated per F1 molecule under conditions where inhibition of ATPase activity is linearly related to iodine incorporation. The molecular size of F1 after iodination is unchanged, indicating that inactivation is due to modification of essential amino acid residues rather than subunit dissociation. Treatment of F1, with 20-50 microM [125I]iodine followed sequentially by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the beta subunit is preferentially labeled. Significantly, about two atoms of iodine per beta subunit are incorporated. Some iodine amounting to less than 23% of the total radioactivity placed on the gels is recovered in the alpha and gamma subunits whereas no radioactivity is detected in the delta and epsilon subunits. Iodination of F1 appears to modify essential residues other than those involved in substrate or product binding per se. Thus, nucleotide binding to F1 is unaltered by iodine, and neither phosphate, MgADP, nor MgATP protects F1 against inhibition by this agent.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Mitochondrial ATP synthase complex: interaction of its F1 adenosinetriphosphatase moiety with the heavy atom iodine. 288 83

The ATP-hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X-100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1 F0 type. The ATPase was specifically activated by Na+ ions yielding a 15-fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X-100 caused a further 1.5-fold activation. In the absence of Na+ Triton stimulated the ATPase about 13-fold. The Triton-stimulated ATPase was further activated about 1.5-2-fold by Na+ addition. The ATPase extracted by the low-ionic-strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1-ATPase subunit structure with Mr values of 58,000 (alpha), 56,000 (beta), 37,600 (gamma), 22,700 (delta), and 14,000 (epsilon). The F1-ATPase was not stimulated by Na+ ions. The membrane-bound ATPase was reconstituted from the purified F1 part and F1-depleted membranes, thus further indicating an F1 F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane-bound F0 part of the enzyme complex.
...
PMID:Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type. 288 96

Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.
...
PMID:F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane. 289 Mar 75

A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation.
...
PMID:Cross-linking study of the quaternary fine structure of mitochondrial F1-ATPase. 289 Jun 32


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---